PROJECT SUMMARY/ABSTRACT: The Mediator protein complex functions as a coactivator of RNA Polymerase II (Pol II) transcription in eukaryotic cells. Mediator interfaces the interaction between Pol II and transcription factors to efficiently regulate gene expression. It is critical for integrating the signal of transcription factors bound at cis-regulatory chromatin elements (enhancers) that are believed to loop to active gene loci. Misregulation of gene expression leads to disease, including cancer and developmental disorders. Yet, the organization of Mediator and Pol II, and the mechanism by which they interact to regulate gene expression is unknown to date. Our long-term goal is to dissect the biophysical mechanisms and principles underlying the highly-regulated control of gene expression in eukaryotic cells. The objective of this grant is to characterize the spatiotemporal organization of Mediator and Pol II in live cells. The central hypothesis is that Mediator and Pol II form functional clusters with other transcription factors at enhancer DNA elements, then those enhancer associated clusters can dynamically associate with promoter DNA to activate gene expression. Our rationale is that direct measurements of Mediator and Pol II interaction will provide important insight into how expression of genes in eukaryotic cells is regulated. Our specific aims will test the following hypotheses: (Aim 1) to determine the mechanisms by which condensates of Mediator and Pol II affect gene expression (Aim 2) to determine the dynamics and functional relevance of the transient population of Pol II and Mediator clusters for gene expression in living cells (Aim 3) to determine how changes in cluster composition result in different functional and Dynamic Properties. Upon conclusion, we will understand the mechanisms by which Mediator and Pol II clusters, both the persistent condensates (~10% of cluster population in stem cells) and transient clusters (90% of the population in stem cells), act to regulate gene expression in living cells. This contribution is significant since it will lead to a new paradigm for describing how transcription is regulated in eukaryotic cells with important implications for development and disease. The proposed research is innovative because we investigate the spatiotemporal organization and mechanism of interaction between Mediator and Pol II at the single-cell level and with super-resolution methods. Insight into how clustering affect transcription is impactful as it provides new avenues for understanding how long-range enhancer-promoter interaction dynamically control of gene expression, and thus how its misregulation may occur in disease.